RESUMO
The regulation of homologous recombination (HR) by p53 has been currently associated with its nontransactivating function; now the transcriptional repression of the Rad51 gene by p53 has been elucidated. This extra control by p53 involves the transcriptional downregulation of Rad51 protein, which in turn accounts for the reduction of functional Rad51 foci. Down regulation of Rad51 protein might be relevant, considering that Rad51 is a rate-limiting factor in HR. Moreover, impaired Rad51-repression by p53 mutant proteins may contribute to malignant transformation.
Assuntos
Rad51 Recombinase/metabolismo , Recombinação Genética , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Regulação para Baixo , Humanos , Mutação , Rad51 Recombinase/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
DNA repair by homologous recombination is involved in maintaining genome stability. Previous data report that wild-type p53 suppresses homologous recombination and physically interacts with Rad51. Here, we show the in vivo binding of wild-type p53 to a p53 response element in the promoter of Rad51 and the downregulation of Rad51 messenger RNA and protein by wild-type p53, favoured by DNA damage. Moreover, wild-type p53 inhibits Rad51 foci formation in response to double-strand breaks, whereas p53 contact mutant R280K fails to repress Rad51 mRNA and protein expression and Rad51 foci formation. We propose that transcriptional repression of Rad51 by p53 participates in regulating homologous recombination, and impaired Rad51 repression by p53 mutants may contribute to malignant transformation.
Assuntos
Rad51 Recombinase/metabolismo , Recombinação Genética , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Técnica Indireta de Fluorescência para Anticorpo , Corantes Fluorescentes , Genes Reporter , Humanos , Luciferases/análise , Luciferases/metabolismo , Microscopia de Fluorescência , RNA Mensageiro/metabolismo , Rad51 Recombinase/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Prolactin (PRL) stimulates breast cancer cell proliferation; however, the involvement of PRL-activated signaling molecules in cell proliferation is not fully established. Here we studied the role of c-Src on PRL-stimulated proliferation of T47D and MCF7 breast cancer cells. We initially observed that PRL-dependent activation of focal adhesion kinase (Fak), Erk1/2, and cell proliferation was mediated by c-Src in T47D cells, because expression of a dominant-negative form of c-Src (SrcDM, K295A/Y527F) blocked the PRL-dependent effects. The Src inhibitor PP1 abrogated PRL-dependent in vivo activation of Fak, Erk1/2, p70S6K, and Akt and the proliferation of T47D and MCF7 cells; Janus kinase 2 (Jak2) activation was not affected. However, in vitro, Fak and Jak2 kinases were not directly inhibited by PP1, demonstrating the effect of PP1 on c-Src kinase as an upstream activator of Fak. Expression of Fak mutant Y397F abrogated PRL-dependent activation of Fak, Erk1/2, and thymidine incorporation, but had no effect on p70S6K and Akt kinases. MAPK kinase 1/2 (Mek1/2) inhibitor PD184352 blocked PRL-induced stimulation of Erk1/2 and cell proliferation; however, p70S6K and Akt activation were unaffected. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished cell proliferation and activation of p70S6K and Akt; however, PRL-dependent activation of Erk1/2 was not modified. Moreover, we show that both c-Src/PI3K and c-Src/Fak/Erk1/2 pathways are involved in the up-regulation of c-myc and cyclin d1 expression mediated by PRL. The previous findings suggest the existence of two PRL-dependent signaling cascades, initiated by the c-Src-mediated activation of Fak/Erk1/2 and PI3K pathways that, subsequently, control the expression of c-Myc and cyclin D1 and the proliferation of T47D and MCF7 breast cancer cells.